Neurotransmitter transporters constitute an important class of related proteins, whose major function is the re-uptake of molecules released upon fusion of synaptic vesicles, thus aiding the rapid clearance of the neurotransmitter from the synaptic cleft. In dopaminergic neurons, the dopamine transporter (DAT) has long been implicated in the effects of numerous psychostimulants and substances of abuse, such as amphetamines and cocaine, which induce an accumulation of dopamine (DA) in the extracellular space. The normal transport cycle of the DAT is electrogenic and coupled to the stoichiometric movement of Na and CI ions, whose electrochemical potential can fuel the translocation of DA against its concentration gradient. This process is reversible. Recent observations have demonstrated that, in the substantia nigra (SN), activation of neural inputs can lead to the release of DA through reverse transport. Such mechanisms could enable the release of DA from non-synaptic regions, a concept of far-reaching implications. This proposal will exploit a new model system, developed in native dopaminergic neurons, which affords a great degree of experimental control together with the high resolution needed for single-cell detection of DA release. The goals are to identify the physiological determinants for DAT reversal during neuronal activity (aim 1) or exposure to amphetamines (aim 2). Using amperometric and patch-clamp recordings of isolated dopaminergic neurons from the SN, we shall first identify the variables (membrane potential, ionic currents, local Na gradients...) affected by the release paradigms and, conversely, measure DA release while manipulating these variables, in order to determine their individual contribution to DAT reversal. Should the observations depart from the predicted behavior of ion-coupled DAT cycling (as suggested for amphetamine by preliminary studies), we shall examine whether neuronal activity and amphetamine recruit distinct modes for DAT reverse operation. Finally, to link these studies to the releasing function of DAT in a physiological context, we shall use multiphoton imaging of fluorescent Na indicators, in dopaminergic neurons maintained in slices, to determine how neural excitation and amphetamines affect local Na gradients (aim 3). These experiments may identify new mechanisms by which mid-brain dopaminergic systems are utilized for ceil-cell communication and are targeted by psychostimulant drugs.